Fatty acids are important components of lipids such as phospholipids and triacylglycerols. Fatty acids containing two or more unsaturated bonds, which are collectively referred to as polyunsaturated fatty acids (PUFAs), and are known to include arachidonic acid, dihomo-γ-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. Various physiological activities have been reported for these fatty acids (non-patent document 1).
Among them, arachidonic acid has attracted attention as an intermediate metabolite in the synthesis of prostaglandins, leukotrienes and the like, and many attempts have been made to apply it as a material for functional foods and medicines. Furthermore, arachidonic acid is contained in breast milk so that it is important for the growth of infants, especially for the growth of fetal length and brain, and therefore, it also attracts attention as well as DHA (docosahexaenoic acid) in a nutritional aspect as a necessary component for the growth of infants.
These polyunsaturated fatty acids are expected to be applied in various fields, but some of them cannot be synthesized in vivo in animals. This has led to development of methods for obtaining polyunsaturated fatty acids by culturing various microorganisms. Attempts to produce polyunsaturated fatty acids in plants have also been made. In such cases, polyunsaturated fatty acids are known to be accumulated as components of reserve lipids such as triacylglycerols, for example, in microbial cells or plant seeds.
Although the molecular structures of enzymes involved in de novo fatty acid synthesis and fatty acid chain elongation differ between prokaryotes and eukaryotes, the mechanisms of enzymatic reactions are similar in any type of cells. Fatty acid biosynthesis starts from acetyl-CoA, and maronyl-CoA is produced from acetyl-CoA by catalysis of acetyl-CoA carboxylase (E.C.6.4.1.2). Various saturated fatty acids are synthesized by adding two carbon atoms via decarboxylative coupling of acetyl-CoA with malonyl-CoA in a series of condensation-reduction-dehydration-reduction reactions catalyzed by fatty acid synthetases (FASs). Similarly, fatty acid chain elongation reactions involve adding two carbon atoms via decarboxylative coupling of acyl-CoA with malonyl-CoA in a series of condensation-reduction-dehydration-reduction reactions.
Acetyl-CoA carboxylases (hereinafter also referred to as “ACCs”) have been hitherto reported in several organisms. Mammalian ACCs are typical allosteric enzymes having the property of being activated by citric acid, inhibited by long-chain fatty acid CoA esters and inactivated by phosphorylation. In fungi, the ACC from yeast (Saccharomyces cerevisiae) has been extensively studied.
The ACC from S. cerevisiae is localized in the cytoplasm and mitochondria and encoded by the ACC1 and HFA1 genes, respectively. The ACC1 gene is known to be an essential gene whose deletion leads to death (non-patent document 2). Analysis of variant strains revealed that the ACC1 gene is also involved in the transport of polyA+ mRNA from the nucleus and other roles (non-patent document 3).
In plants, attempts were made to increase fats in seeds using ACC genes (non-patent document 4). For example, a report shows that the fatty acid content on a dry weight basis increased and compositional ratio of the fatty acids also changed in the seeds of transgenic Brassica napus L. expressing the ACC of Arabidopsis thaliana (non-patent document 5). However, the pattern of change in compositional ratio of fatty acids depends on the compositional ratio of fatty acids inherent in the host organism and the ACC gene transduced. On the other hand, ACC activity undergoes various regulations not only at the expression level but also at the protein level (non-patent documents 3 and 4), and it is also influenced by interactions with other enzymatic proteins functioning in a series of fatty acid synthesis systems. Therefore, a suitable ACC gene may be necessary to obtain a desired fatty acid composition depending on the host organism to be transformed.
As for the ACC gene of a lipid-producing fungus Mortierella alpina (hereinafter also referred to as “M. alpina”), a fragment of a gene for an ACC homolog from strain CBS 528.72 presumably having ACC activity has previously been known (non-patent document 6). However, it has not been confirmed yet that a protein having this fragment has ACC activity. M. alpina strain CBS696.70 has been assessed for fat accumulation and acetyl-CoA carboxylase activity (non-patent document 7).